Abstract: Objective To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. Methods We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). Results In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis (P<0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 (P<0.01) and Bax (P<0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 (P<0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. Conclusion ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.